Monosaccharide analog-based glycosidase inhibitors

ABSTRACT

Broad spectrum glycosidase inhibitors are produced from monosaccharide lactams by conversion to amidines, amidrazones, or amidoximes. The inhibitors have the general formula: ##STR1## wherein the N and C are joined as part of a monosaccharide azaanalog ring and wherein --NRQ, together with the nitrogen and carbon atoms, form a chemical group selected from amidines, amidrazones, and amidoximes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 07/453,051,filed Dec. 13, 1989, now abandoned which is a continuation of U.S. Ser.No. 07/106,486, filed Oct. 6, 1987, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 06/799,708, filed Nov. 19, 1985,now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to amidine, amidrazone and amidoxime derivativesof monosaccharides which have an unexpectedly broad spectrum glycosidaseinhibitory effect. The invention further relates to methods of preparingthe derivatives, novel intermediates, methods of preparing theintermediates, and the use of the derivatives to inhibit glycosidases,particularly multiple classes thereof.

Glycosidases are enzymes that catalyze the hydrolysis of glycosidicbonds and are essential for the normal growth and development of allorganisms. Their vital role is reflected in their wide distribution innature. They participate in biologically significant reactions such asthe breakdown of carbohydrate foodstuffs, the processing of eucaryoticglycoproteins, the catabolism of polysaccharides and glycoconjugates,and the like. Certain glycosidase inhibitors have been found to inhibithuman immunodeficiency virus (HIV) syncytium formation and virusreplication, thereby indicating their potential use as antiretroviralagents. And some glycosidases are showing promise in treating diabetesor as antiviral and anticancer agents.

Polyhydroxylated piperidines constitute a class of naturally-occurringglycosidase inhibitors. Examples of such monosaccharide analogs whichcontain a nitrogen atom (an endocyclic nitrogen) in place of thepyranose oxygen include: nojirimycin (A) and 1-deoxynojirimycin (B)(Inouye et al., Tetrahedron, 1968, 24, 21252144), 1-deoxymannojirimycin(C) (Fellows et al, J. Chem. Soc. Chem. Commun., 1979, 977-8), andgalactostatin (D) (Miyake et al., Agric. Biol. Chem., 1988, 52, 661-6).These alkaloids are quite specific inhibitors of their targeted enzymes.The arrangement of the hydroxyl groups apparently determines individualenzyme specificity of these inhibitors. Compounds A and B are potentglucosidase inhibitors that closely resemble glucose; compound Cinhibits mannosidases; and compound D inhibits galactosidases. Theinhibitors are believed to function by mimicking the natural substrates.Compound B has been shown to interfere with the infectivity of HIV(Gruters et al., Nature, 1987, 330, 74-7).

Since a number of glycosidases are insensitive to the naturallyoccurring alkaloids, tremendous effort has been devoted to thedevelopment of synthetic inhibitors which may inhibit those enzymes.Examples include inhibitors of: jack bean α-mannosidase (E) (Eis et al.,Tetrahedron Lett, 1985, 26, 5397-8), coffee bean α-galactosidase (F)(Bernotas et al., Carbohydr. Res., 1987, 167, 305-11),α-N-acetylglucoaminidase (G) (Fleet et al., Chem. Lett., 1986, 1051-4),and α-hexosaminidase (H) (Bernotas et al., Carbohydr. Res., 1987, 167,312-6). In these polyhydroxylated piperidines, the ring oxygen in thesugar has been replaced by a nitrogen to form azasugars. Unfortunately,the analogy of using azasugars as glycosidase inhibitors is not alwaysapplicable. For example, while polyhydroxylated pyrrolidines are betterinhibitors of yeast α-glucosidase, their relative activities arereversed for a number of mouse gut disaccharides. Therefore, the actualeffectiveness of a given compound as a glycosidase inhibitor of aspecific enzyme and the effect on specificity by a given structuralchange of the compound remain unpredictable.

Other potent glycosidase inhibitors contain at the anomeric position anexocyclic nitrogen. (Lai et al., Biochem. Biophys. Res. Commun., 1973,54, 463-8) For example, glucosylamine inhibits both α- andβ-glucosidases.

The present invention is the result of attempting to combine into asingle compound several specific features which, among many others, havebeen found to be present in some glycosidase inhibitors to determinewhether the combination compound would prove beneficial. Thus, thecompounds of this invention contain both endocyclic and exocyclicnitrogens in an sp² -hybridized functional group and also have aflattened, half-chair conformation. While previous glycosidaseinhibitors have generally shown activity only against a single class ofglycosidases, i.e. glucosidases, mannosidases, galactosidases, etc.,they have not demonstrated broad spectrum activity across multipleclasses. The amidine, amidrazone, and amidoxime derivatives of themonosaccharide analogs of the present invention, on the other hand, havebeen unexpectedly found to exhibit an apparently unique and broadspectrum of glycosidase inhibitory activity which extends across classlines, i.e. independent of the sugar analog from which they are formed.

Previous attempts at preparing amidine derivatives of glucose to formpossible glycosidase inhibitors were reported by Bird et al., Can. J.Chem., 1990, 68(2), 317-22. Bird et al. started with glucose andprepared 5-azido-2,3,4,6-tetra-O-benzyl-5-deoxy-D-gluconitrile and2,3,4,6-tetra-O-benzyl-5-deoxy-5-trifluoroacetamido-D-gluconitrile, butwas unable to convert these compounds into a protected5-amino-5-deoxy-D-gluconitrile and to subsequently cyclize them to anamidine analog of glucose.

Accordingly, it is an object of the present invention to produceglycosidase inhibitors which combine both endocyclic and exocyclicnitrogens into an sp² -hybridized functional group and also have ahalf-chair conformation.

It is a further object to produce novel amidine, amidrazone, andamidoxime derivatives of monosaccharides.

It is a further object to develop a process for producing glycosidaseinhibiting compounds which process could generate amidines, amidrazonesor amidoximes of different glycoses by simply changing the configurationof the starting material.

These and still further objects will be apparent from the followingdetailed description of the invention.

SUMMARY OF THE INVENTION

In accordance with the present invention, novel amidine, amidrazone, andamidoxime derivatives of monosaccharides, i.e. hexoses and pentoses, aresynthesized from appropriate thionolactam precursors. The thionolactamprecursors are themselves novel compounds and are prepared by the actionof Lawesson's reagent on the appropriate lactams in which the hydroxylgroups have been previously protected/blocked.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a and b are the graphs of (a) 1/V vs 1/[S] and (b) L-B slopes vs[I] for the D-glucoamidine of Example 1 against almond β-glucosidase.

FIGS. 2aand b are the graphs of (a) 1/V vs 1/[S] and (b) L-B slopes vs[I] for the D-glucoamidine of Example 1 against jackbeanalpha-mannosidase.

FIGS. 3a and b are the graphs of (a) 1/V vs 1/[S] and (b) L-B slopes vs[I] for the D-glucodimethylamidine of Example 2 against almondβ-glucosidase.

FIGS. 4a and b are the graphs of (a) Hanes-Woolf Plot and (b)Y-intercepts from Hanes-Woolf Treatment plotted vs. [I] for theD-glucodimethylamidine of Example 2 against almond β-glucosidase.

FIGS. 5a and b are the graphs of (a) 1/V vs 1/[S] and (b) L-B slopes vs[I] for the D-glucoamidrazone of Example 8 against almond β-glucosidase.

FIGS. 6a and b are the graphs of (a) 1/V vs 1/[S] and (b) L-B slopes vs[I] for the D-glucoamidrazone of Example 8 against jackbeanalpha-mannosidase.

FIGS. 7a and b are the graphs of (a) 1/V vs 1/[S] and (b) L-B slopes vs[I] for the D-glucoamidrazone of Example 8 against bovineβ-galactosidase.

FIGS. 8a and b are the graphs of (a) 1/V vs 1/[S] and (b) L-B slopes vs[I] for the D-mannoamidrazone of Example 9 against almond β-glucosidase.

FIGS. 9a and b are the graphs of (a) 1/V vs 1/[S] and (b) L-B slopes vs[I] for the D-mannoamidrazone of Example 9 against jackbeanalpha-mannosidase.

FIGS. 10a and b are the graphs of (a) 1/V vs 1/[S] and (b) L-B slopes vs[I] for the D-glucoamidoxime of Example 12 against almond β-glucosidase.

FIGS. 11a and b are the graphs of (a) 1/V vs 1/[S] and (b) L-B slopes vs[I] for the D-mannoamidoxime of Example 14 against jackbeanalpha-mannosidase.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The compounds of the present invention combine a halfchair, i.e.flattened anomeric, conformation with both exocyclic and endocyclicnitrogens to form an sp² -hybridized functional group in amonosaccharide which conformation and group produce broad spectrumglycosidase inhibitory activity.

Specific compounds of this invention are derivatives of monosaccharidesand have the general formula: ##STR2## wherein R is selected fromhydrogen, alkyl, substituted alkyl, alkaryl, aryl, substituted aryl,aralkyl, and Q is selected from R, --NR₂, and --OH; wherein when Q is Rthe two R groups may be joined together to form a ring containing atleast 2 o carbon atoms; and wherein the --N═C-- is part of amonosaccharide azaanalog ring.

Preferably, --NRQ together with the carbon atom to which it is attachedand said nitrogen atom form a functional group selected from amidines,amidrazones, and amidoximes.

The amidines have the general formula: ##STR3## wherein each R ishydrogen or a hydrocarbon group. Suitable hydrocarbon groups aregenerally selected from alkyl, substituted alkyl, alkaryl, aryl,substituted aryl, aralkyl, or the two R groups are joined together toform a ring containing at least 2 carbon atoms. Preferably each R ishydrogen or alkyl C₁₋₆.

The amidrazones have the general formula: ##STR4## wherein R¹ and R² areeach hydrogen or a hydrocarbon group. Suitable hydrocarbon groups aregenerally selected from alkyl, substituted alkyl, alkaryl, aryl,substituted aryl, aralkyl, or the two R² groups are joined together toform a ring containing at least 2 carbon atoms.

The amidoximes have the general formula: ##STR5## wherein R³ is hydrogenor a hydrocarbon group. Suitable hydrocarbon groups are generallyselected from alkyl, substituted alkyl, alkaryl, aryl, substituted aryl,and aralkyl.

Preferred numbers of carbon atoms and substituents for the varioushydrocarbon groups in each of the above formulae include: alkyl from 1to about 18 carbon atoms, substituted alkyl from about 1 to 18 carbonatoms and wherein the substituents do not substantially interfere withthe glycosidase inhibitory action of the compounds, alkaryl from 7 toabout 18 carbon atoms and which may also be substituted, aryl from 6 to18 carbon atoms, substituted aryl from 6 to about 18 carbon atoms andwherein the substituents do not substantially interfere with theglycosidase inhibitory action of the compounds, aralkyl from 7 to about18 carbon atoms. Specific examples of useful substituents include:hydroxypropyl, hydroxybutyl, naphthyl, 2-furyl, imidazolyl,p-methoxyphenyl, p-fluorophenyl, shingosinyl, phenacylmethyl, and thelike.

The terminal N of the amidrazones is reactive and thus, either before orafter the formation of the amidrazone, it may be reacted with acidhalides, anhydrides, esters, isocyanates, isothiocyanates, and the likein a conventional manner. Similarly, the --OH group which is part of theamidoxime functionality of the amidoximes (as opposed to themonosaccharide hydroxyls) is reactive and may be etherified oresterified either before or after formation of the amidoxime.

In the azaanalog derivatives of monosaccharides of this invention, the----N═C---- portion of the hexose or pentose ring (in which the usualoxygen atom of a monosaccharide has been replaced by a nitrogen atom)also forms part of the amidine, amidrazone, or amidoxime functionalgroup.

Monosaccharides on which the present analog derivatives are based arehexoses or pentoses which form cyclic structures. Suitablemonosaccharides include the glucose and ribose families as well as thestereoisomers, deoxy and substituted derivatives thereof. Specificmonosaccharides upon which the azaanalog derivatives of this inventionmay be based include, for example, (i) aldohexoses: altrose, allose,mannose, glucose, galactose, talose, gulose, idose, fucose,2-deoxy-2-aminoglucose, 2-deoxy-2-aminogalactose, and the like; (ii)aldopentoses: ribose, arabinose, 2-deoxyribose, 2-deoxyarabinose, andthe like. Preferred such monosaccharides are mannose, glucose,galactose, fucose, 2-deoxy-2-aminoglucose, 2-deoxy-2-aminogalactose,ribose, arabinose, 2-deoxyribose, and 2-deoxyarabinose. The currentlymost preferred monosaccharides are glucose, mannose, and galactose dueto the ready availability of lactams thereof which are startingmaterials for the preparative part of this invention.

The preparation of the compounds of the invention will be describedherein commencing from the lactams of a monosaccharide. Lactams areorganic compounds which contain an --NH--CO-- group within a ring. Theyare generally formed by the elimination of water from neighboringcarboxyl and amino groups. The monosaccharide lactams of the presentinvention are therefore cyclic amides having the general structure:##STR6## The reactions used herein to convert a lactam to thecorresponding amidines, amidrazones, and amidoximes of the invention areindependent of both the stereochemistry and the size of the balance ofthe monosaccharide ring. Thus the present invention is applicable to anymonosaccharide. Numerous references to the preparation of lactams ofmonosaccharides have been reported in the recent chemical literature.The references include: Inouye et al., Tetrahedron, 1968, 24(5),2125-44; Hanessian, J. Org. Chem., 1969, 34(3), 675-81; Ito et al., JP46-024,382, 1971; Tsuruoka et al., Meiji Seika Kenkyu Nempo, 1973, No.13, 80-4; Niwa et al., U.S. Pat. No. 3,956,337; Tsuruoka et al., JP50-129,572; Niwa et al., J. Antibiot., 1984, 37(12), 1579-86; Ehata etal., JP 61-280,472; Miyake et al., Agric. Biol. Chem., 1988, 52(3),661-6; Shing, J. Chem. Soc. Chem. Commun., 1988, 18, 1221; Fleet et al.,Tetrahedron, 1989, 45(1) 319-26;Tsuruoka et al., JP 63-258,421 and JP63-216,867; Fleet et al., Tetrahedron Lett., 1990, 31(3), 409-12.

A particularly suitable procedure for preparing D-gluconolactam, i.e.the lactam of D-glucose, by the oxidation of nojirimycin is described inExample 1 below. This procedure represents an improved oxidationprocedure over previous procedures in that the yield obtained issignificantly higher than the published yield, i.e. about 50% vs. about30%.

Other monosaccharide lactams may be prepared in accordance with one ormore of the preparative procedures cited above.

The hydroxyl groups of the monosaccharide lactams are then protectedfrom reaction with Lawesson's reagent which is subsequently used to formthionolactams which are then reacted with NH-containing compounds toyield the desired functional compounds. Suitable blocking/protectinggroups are those that, for a particular lactam, do not participate inneighboring group interactions which result in the formation ofundesired compounds. Preferably, the groups will also be removable underrelatively mild conditions. Examples of such suitable groups includetrimethylsilyl, acetyl, t-butyldimethylsilyl, trityl, methoxymethyl,benzyl, and benzoyl.

The protecting groups may be incorporated onto the lactams by anyconventional suitable manner. For example, trimethylsilyl groups may beproduced by suspending the lactam in a non-solvent, such as drypyridine; adding a mixture of chlorotrimethylsilane andhexamethyldisilazane; stirring at room temperature; and then recoveringthe resulting persilylated lactam. Acetyl groups may be provided byperacetylation with Ac₂ O, NaOAc, at room temperature for extendedperiods.

The protected hydroxyl group-containing lactam is then converted to thecorresponding thionolactam by reaction with Lawesson's reagent(p-methoxyphenylthionophosphine sulfide dimer) in a conventional manner,i the protected lactam in a solvent such as benzene under an inert gassuch as argon at room temperature and then heating to reflux. Lawesson'sreagent and its use are described in Scheibye et al., Bull. Soc. Chem.Belg., 1978, 87, 229-38. The reaction with Lawesson's reagent convertsthe C═O of the lactam to a C═S of the thionolactam without disturbingthe protecting groups.

The protecting groups are then removed in a conventional manner such asby acidic hydrolysis. Any sufficiently strong acid which does not effectthe balance of the compounds may be used. Suitable such acids includehydrochloric acid, sulfuric acid, perchloric acid, fluoroboric acid,p-toluenesulfonic acid, and the like. Other methods of removingprotecting groups include hydrogenolysis, alcoholysis, and fluoridetreatment. Alternatively, when the final products are sufficientlystable the protecting groups may be allowed to remain on thethionolactam during reaction with the NH-compound and then removed asdiscussed.

The thionolactams of the monosaccharides produced are believed novelcompounds themselves. They are useful intermediates in the preparationof the glycosidase inhibitors. Specifically, depending upon theparticular NH-containing compound with which they are reacted, they mayyield amidine, amidrazone, and amidoxime derivatives.

Thus, the glycosidase inhibitor compounds of the present invention areultimately synthesized by reaction of monosaccharide thionolactamprecursors of the general formula: ##STR7## with an NH-containingcompound, preferably in solution. Suitable solvents include water,alcohols, acetonitrile, and the like. Preferably the solvent ismethanol, ethanol,isopropyl alcohol or acetonitrile. Suitabletemperatures will range from about 0° C. to reflux.

The amidine derivatives are readily prepared by reacting thethionolactam with ammonia or the appropriate mono- or disubstitutedamine. Suitable such amines or ammonia are those of the formula NHR₂wherein each R is independently selected from hydrogen, alkyl,substituted alkyl, alkaryl, aryl, substituted aryl, aralkyl, or the twoR groups are joined together to form a ring contain at least 2 andgenerally less than about 8 carbon atoms.

The amidrazone derivatives are prepared in like manner to the amidinesbut with the thionolactam being reacted with hydrazine or a hydrazinederivative, generally of the formula NHR¹ -NR² ₂ wherein R¹ and each R²are individually selected from hydrogen, alkyl, substituted alkyl,alkaryl, aryl, substituted aryl, aralkyl, or the two R² groups arejoined together to form a ring contain at least 2 carbon atoms andgenerally less than about 8 carbon atoms. Either before or afterformation of the amidrazone, the terminal N of the hydrazine compoundmay be reacted with an acid halide, anhydride, ester, isocyanate,isothiocyanate or the like.

The amidoxime derivatives are also prepared in like manner to theamidines and amidrazones but with the thionolactam being reacted with ahydroxyamine of the general formula NHR³ OH wherein R³ is selected fromhydrogen, alkyl, substituted alkyl, alkaryl, aryl, substituted aryl, andaralkyl. Either before or after formation of the amidoxime, the OH-groupmay be esterified or etherified by reaction with an acid halide,anhydride, ester, isocyanate, isothiocyanate or the like.

While the degree of glycosidase inhibitory activity on a particularglycosidase enzyme or series of glycosidase enzymes is likely to atleast partially depend upon the specific R groups which are provided onspecific compounds, the principles of this invention have not been founddependent thereon. Preferred numbers of carbon atoms and substituentsfor the various groups in each of the above formulae include: alkyl from1 to about 10 carbon atoms, substituted alkyl from about 1 to 10 carbonatoms and wherein the substituents do not substantially interfere withthe glycosidase inhibitory action of the compounds, alkaryl from 7 toabout 18 carbon atoms and which may also be substituted, aryl from 6 to18 carbon atoms, substituted aryl from 6 to about 18 carbon atoms andwherein the substituents do not substantially interfere with theglycosidase inhibitory action of the compounds, aralkyl from 7 to about18 carbon atoms. Examples of specific substituents include: halogens,alkyl, aryl, alkoxy, fluoro, cyano, carboalkoxy, reto, thio, nitro, andthe like.

The glycosidase inhibitors of this invention are generally useful with abroad spectrum of glycosidase enzyme classes as opposed to a singleclass. While the general inhibitory strength of a particular compoundwill vary depending upon its particular structure and the enzyme, thecompounds of this invention have exhibited potent inhibitory effectsagainst glucosidases, mannosidases, and galactosidases. Other classes ofglycosidase enzymes for which the inhibitors are likely to be usefulinclude one or more of: L-fucosidases, sialidases, glucosaminidases,chitinases, lysozyme, cellulases, and the like. Specific glycosidaseenzymes which have been inhibited by one or more of the claimedcompounds include yeast α-glucosidase, aspergillus nigeramyloglucosidase, almond β-glucosidase, jackbean α-mannosidase, greencoffee bean α-galactosidase, and bovine liver β-galactosidase.

While the D-glucoamidines have been found to be potent inhibitorsagainst gluco, manno and galactosidases, they have also proved to beunexpectedly labile, undergoing rapid hydrolysis above pH 7. Theamidrazone and amidoxime derivatives are much more stable and they wereunexpectedly found to be generally even more potent inhibitors, furtheremphasizing the importance of binding interactions with the anomericregion of the glycosyl cation. The amidoximes represent the firstnear-neutral monosaccharide analogs possessing the half-chairconformation of the glycosyl intermediates.

GENERAL EXPERIMENTAL INFORMATION

Proton NMR spectra were taken on a Bruker WM-300 spectrometer. Allchemical shifts were reported on the δ scale in parts per milliondownfield from Me₄ Si. Spectra taken in CDCl₃ were referenced to eitherMe₄ Si (0.00 for compounds with aromatic protons) or residual CHCl₃(7.24, for compounds without aromatic protons). Spectra taken in D₂ Owere referenced to HOD (4.67), and those in CD₃ OD were referenced toCHD₂ OD (3.30). Carbon-13 NMR spectra were taken on a Varian XL-400 (100MHz), Bruker WM-300 (75 MHz) spectrometer and referenced to p-dioxane(66.5 ppm) or CH₃ OH (49.0 ppm). Infrared spectra were taken on aMattson Galaxy Model infrared spectometer. Ultraviolet absorptionspectra were measured on a Hewlett-Packard HP 8451 A Diode ArraySpectrometer. Mass spectra were obtained from a Finnigan 3300 massspectrometer. Chemical ionization spectra were obtained using isobutaneas reagent gas; electron impact spectra were run at 70 e V ionizingvoltage. Fast atom bombardment spectra were obtained in glycerol matrixon a Kratos MS-890 Spectrometer. Optical rotations were measured on aPerkin Elmer 24 polarimeter. Sample concentrations were expressed ingrams of sample per 100 cc of solvent. High performance liquidchromatography was performed with an Eldex 9600 system using a μ-Porasilcolumn (internal diameter 7.8 mm).

Anhydrous methanol was prepared by distillation from Mg(OCH₃)₂. Benzene,pyridine, chlorotrimethylsilane and hexamethyldisilane were dried anddistilled from CaH₂. All enzymes were obtained from Sigma and used asit.

In the following non-limiting examples, all parts and percents are byweight unless otherwise specified. Also, the compounds produced weregenerally isolated as their quaternary acetate salts, though other saltsmay be substituted in a conventional manner.

EXAMPLE 1 Preparation of Glucoamidine Oxidation of (+)-Nojirimycin to(3R,4S,5R,6R)-6-Hydroxymethyl-3,4,5-trihydroxy-2-piperidone[D-(+)-Gluconolactam

To a stirred suspension of nojirimycin bisulfite addition product (1.02g, 4.22 mmol; Baeyer AG) in distilled water (25 mL) was added activatedDowex 1×2-200 resin (HO-form, 10 g, Aldrich) to make the pH 8-10. Afterstirring 30 min at room temperature, the resin was filtered, rinsed withdistilled water (160 mL) and the combined filtrates lyophilized toafford a crude sample of (+)-nojirimycin (0.89 g, R_(f) 0.58 in 4:1ethanol:H₂ O) which was dissolved in distilled water (15 mL) and usedimmediately in the next step.

The magnetically stirred aqueous nojirimycin solution was treated withalternating portions of 0.1M I₂ -0.5M KI solution (83 mL; 2 mL aliquots)and 0.1M NaOH (100 mL, 2.5 mL aliquots) at room temperature slowly overa period of 90 min. After 24 hours, the brown solution was decolorizedby addition of aqueous NaHSO₃ (1M, 4 mL), then Amberlite IR-120 (H+)resin was added (0.5 g, Aldrich) to bring the pH to 1. After stirring 4hr at room temperature, the resin was filtered and rinsed with water (50mL). The combined filtrates were then neutralized with Dowex MWA-1(Serva Corp.), the resin filtered and washed, and the combined filtrates(ca. 400 mL) concentrated at the rotary evaporator to afford the crudelactam as a white solid. A small portion was chromatographed andrecrystallized from ethanol:water to afford pure lactam whose mp (203°C.) and chiroptical properties were identical with published values.

Silylation of Lactam

Crude (5-6 g) was suspended in dry pyridine (20 mL), then(trimethylsilyl)₂ NH(5 mL) and trimethylsilylchloride (3 mL) was addedand the dark brown reaction mixture stirred at room temperature for 90minutes. Concentration in vacuo afforded a brownish foam (1.6 g) whichwas flash chromatographed on SiO₂ (40 mm×4.5 inch column; 7:1hexanes:ethyl acetate; 8 mL fractions collected) to furnish thepersilylated gluconolactam (1.14 g, 2.46 mmol, 50%): Rf 0.27 (1:7hexanes:ethyl acetate); [α]_(D) +71° (c=0.54, CHCl₃); ¹ H-NMR δ (CDCl₃)5.88 (br. s, 1H), 3.87 (d, 1H, J=8.6 Hz), 3.78 dd, 1 H, J=8.3, 1.8 Hz),3.69 (dd, 1 H, J=8.7, 8.6 Hz), 3.43 (dd, 1 H, J=8.4, 8.4 Hz), 3.35-3,25(m, 2H), 0.18 (s, 9 H), 0.14 (s, 18 H), 0.09 (s, 9 H); ¹³ C-NMR (CDCl₃)170.9, 76.2, 74.0, 71.4, 63.7, 57.0, 0.91, 0.73, -0.70; IR (film) 3210,3110, 2980, 2905, 1690, 1320, 1250, 1130, 950, 840 cm⁻¹ ; CIMS (methane)m/e 478 (M+2, 65%) 466 (M+1, 50%) 450 (M+-CH₃, 100%).

Formation of D-Glucothionolactam(3R,4S,5R,6R)-6-Hydroxymethyl-3,4,5-trihydroxy-2-thionopiperidone

To a solution of the persilylated lactam of above (0.284 g, 0.61 mmol)in benzene (15 mL) under argon at room temperature was added Lawesson'sreagent (p-methoxyphenylthionophosphine sulfide dimer, 0.148 g, 0.6equiv) and the suspension was warmed. At 65° C. the reaction becamehomogeneous and was brought to reflux for 30 min. After concentrating invacuo, the residue was dissolved in CH₃ OH (16 mL), acidified (1:9 conc.HCl:CH30H, 10 drops) and stirred for 35 minutes at room temperature.Concentration afforded a white solid (0.25 g) which was flashchromatographed over SiO₂ (20 mm×6 inch column; 7:3:1 CH₂ Cl₂ CH₃ OH:NH₄OH, 3 mL fractions) to afford slightly impure glucothionolactam (0.105g) which was further purified as follows.

The thionolactam (0.105 g) was dissolved in distilled water (10 mL) andstirred with Norit A (1.0 g) for 30 minutes. The Norit was filtered,rinsed with water (10 mL, discarded), then eluted with 1:1 ethanol:H₂ Oand the eluant concentrated in vacuo to give analytically purethionolactam (74 mg, 0.38 mmol, 63%): R_(f) 0.33 (7:3:1 CH₂ Cl₂ :CH₃OH:NH₄ OH) mp 126°-128° C.; [α]_(D) +31° (c=0.72, CH₃ OH) ¹ H-NMR δ (D₂O) 3.90 (d, 1 H, J=9.5 Hz) 3.78 (dd, 1 H, J=12.3, 2.4 Hz), 3.74 (dd, 1H, J=9.8, 8.7 Hz), 3.67 (dd, 1 H, J=12.4, 4.1 HZ), 3.60 (dd, 1 H, J=9.7Hz), 3.33 (m, 1 H); ¹³ C-NMR (D₂ O, acetone ref.) 214.8, 74.2, 72.6,67.4, 61.6, 59.7; IR (KBr) 3360, 2940, 2900, 1660, 1560, 1450, 1292,1075, 1030 cm⁻¹.

High Resolution FAB-MS: Calculated for C₆ H₁₁ NO₄ S: 193.0409, Found:193.0403.

Analysis for C₆ H₁₁ NO₄ S: Calculated: C, 37.29; H, 5.74; N, 7.25; S,16.56, Found C, 36.17; H, 5.87; N, 7.22; S, 13.94 .

Preparation of D-Glucoamidine

Saturated anhydrous NH₃ in methanol (1.5 mL) was added dropwise to astirred solution of the thionolactam (14 mg, 0.072 mmol) in anhydrousCH₃ OH (1 mL) at room temperature under argon. Thin layerchromatographic monitoring indicated that the thionolactam disappearedin 11 hours and two new spots were visible (baseline and R_(f) 0.08 in7:3:1 CH₂ Cl₂ :CH₃ OH:NH₄ OH). The solution was concentrated in vacuo,redissolved in fresh CH₃ OH (2.5 mL) and acidified to pH 3.5 withanhydrous HCl--CH₃ OH (0.5 mL, prepared from 0.3 mL AcCl in 8 mL CH₃OH). Concentration in vacuo afforded a brown oil which was purified bySiO₂ flash chromatography (8 mm×2.5 inch column; 20:4:1 CH₃ CN:H₂O:HOAc, 2 mL fractions) to afford the amidine HOAc (11.6 mg, 68%): R_(f)0.14 (20:4:1 CH₃ CN:H₂ O:HOAc); [α]_(D) +27° (c=0.17, CH₃ OH); ¹ H-NMR δ(D₂ O) 4.24 (d, 1 H, J=9.6 Hz), 3.77-3.56 (m, 4 H), 3.34 (m, 1 H)1.94-1.76 (s, e H), ¹³ C-NMR (d20, dioxane ref), 179.0, 167.5, 72.0,68.2, 67.3, 60.1, 59.3, 21.9; IR (KBr) 3390, 3240, 2930, 1685, 1575,1555, 1415, 1075 cm⁻¹.

High Resolution FAB-MS: Calc. for C₆ H₁₃ N₂ O₄ : 177.0875; Found:177.0875.

EXAMPLE 2 Preparation of D-Gluco-N,N-Dimethylamidine

Anhydrous dimethylamine was bubbled into dry CH₃ OH (5 mL) under argonat 0° C. until the volume of the solution doubled. Four milliliters ofthis solution was transferred by Teflon cannula to the thionolactam ofExample 1 (11 mg, 0.057 mmol) and the resulting solution was stirred 10minutes at 0° C. before warming to room temperature. Thin layerchromatographic monitoring indicated that the thionolactam disappearedin 8 hours and one new spot was visible (R_(f) 0.17 in 20:4:1 CH₃ CN:H₂O:HOAc). The solution was concentrated in vacuo. redissolved in freshCH₃ OH (3 mL) and acidified under argon to pH 3.0 with anhydrousHCl--CH₃ OH (0.4 mL, prepared from 0.3 mL AcCl in 8 mL CH₃ OH). Afterconcentrating in vacuo the residue was purified by SiO₂ flashchromatography (8 mm×2.5 inch column; 20:4:1 CH₃ CN: H₂ O:HOAc, 2 mLfractions) to afford the amidine.HOAc (10.6 mg, 71%): R_(f) 0.21 (20:4:1CH₃ CN:H₂ O:HOAc); [α]_(D) +15° (c=0.57, CH₃ OH); ¹ H-NMR δ (D₂ O) 4.46(d, 1 H, J=7.0 Hz), 3.86 (dd, 1 H, J=11.6, 9.0 Hz), 3.77 (dd, 1 H,J=9.0, 7.0 Hz), 3.71 (dd, 1 H, J=12,2, 3.8 Hz) 3.60 (dd, 1 H, J=11.6,9.0 Hz), 3.45 (m, 1 H), 3.21 (s, 3H), 3.04 (s, 3H); ¹³ C-NMR (D₂ O,dioxane ref) 179.7, 163.2, 74.5, 68.9, 65.9, 58.9, 58.6, 41.7, 39.4,22.2; IR (KBr) 3320, 2925, 1660, 1565, 1420, 1060 cm⁻¹.

High Resolution FAB-MS: Calculated for C₈ H₁₆ N₂ O₄ : 204.1111; Found:204.1110.

EXAMPLE 3 Preparation of D-Mannoamidines

L-gluconolactone was converted to D-mannolactam by the procedure ofFleet (1989). As described in detail in Example 9 below, the hydroxylgroups of the lactam were blocked with trimethylsilyl groups, and thelactam converted to the thionolactam. Then the amidine was formed byreaction of the thionolactam with ammonia in methanol at roomtemperature and the blocking groups removed, all as done in Example 1for D-gluconolactam.

A portion of the D-mannothionolactam is also reacted with diethylamineto produce the corresponding D-manno-N,N-diethylamidine.

EXAMPLE 4 Preparation of D-Galactoamidines

The procedure of Example 1 is repeated to produce two D-galactoamidinesby reacting D-galactothionolactam (produced from galactostatin) in onecase with ammonia and in the other with benzylamine. The resultingcompounds are D-galactoamidine and D-galacto-N-benzylamidine.

EXAMPLE 5 Preparation of L-Idoamidine

The procedure of Example 1 is repeated to produce two L-idoamidines byreacting L-idothionolactam (produced by the procedure of Ito et al., JP46-024382) in one case with ammonia and in the other with diphenylamine.The resulting compounds are L-idoamidine and L-ido-N,N-diphenylamidine.

EXAMPLE 6 Preparation of D-Glucoepiminoamidine

The procedure of Example 1 is repeated to produce D-glucoepiminoamidineby reacting D-glucothionolactam with ethyleneimine in ethanol. Theresulting compound is D-glucoepiminoamidine which has the exocyclicnitrogen atom in a three membered ring with two carbon atoms.

EXAMPLE 7 Preparation of Riboamidines

The basic procedure of Example 1 is repeated to produce twoD-riboamidines by reacting D-ribothionolactam (produced by the procedureof Hanessian, J. Org. Chem., 1969, 34 (3), 675-81) in one case withammonia and in the other with dimethylamine. The resulting compounds areD-riboamidine and D-ribo-N,N-dimethylamidine.

EXAMPLE 8 Preparation of D-Glucoamidrazone

Anhydrous NH₂ NH₂ (70 μL, 2.208 mmol, distilled from NaOH) was addeddropwise to a stirred solution of D-gluconothionolactam (20.5 mg, 0.106mmol) in anhydrous CH₃ OH (3 mL) in an ice-H₂ O bath under Ar. TLCmonitoring (CH₃ CN:OAc:H₂ O 20:1:4) indicated that the thionolactamdisappeared after 90 min. The solution was concentrated in vacuo and theresidue (24 mg) was purified by SiO₂ chromatography (3.5 in×12 mmcolumn; 25:3:1 CH₃ CN:H₂ O:HOAc, 2 mL fractions) to affordD-glucoamidrazone HOAc (19.3 mg, 73%): R_(f) =0.33 (CH₃ CN:AcOH:H₂ O10:1:4) [α]_(D) +15.6° (c=0.45, MeOH); ¹ H-NMR δ (D₂ O) 4.27 (m, 1 H,J=2.3, 6.9 Hz), 3.81 (dd, 1 H, J=2.8, 12.2 Hz), 3.72-3.67 (m, 3 H), 3.41(m, 1 H), 1.78 (s,3 H); ¹³ C-NMR (D₂ I, dioxane ref.), 179.1, 164.4,72.4, 67.7, 60.2, 59.1, 22.2, IR (KBr), 3310, 2920, 1700, 1665, 1560,1410, 1110, 1070, 1020 cm⁻¹.

High Resolution FAB-MS: Calc. for C₆ H₁₄ O₄ N₃ : 192.0984; Found:192.0989.

EXAMPLE 9 Preparation of D-Mannoamidrazone Preparation ofD-Mannonothionolactam

D-Mannolactam (0.203 c, 1.14 mmol) was dissolved in dry pyridine (15mL), then (TMS)₂ NH (5 mL) and TMSCl (2.5 mL) were added dropwise. Theresulting suspension was stirred at room temperature under Ar for 90min. Concentration in vacuo afforded a white residue (613 mg) which wastriturated with hexanes (60 mL in 3 mL fractions). The combinedtriturants were concentrated using a rotary evaporator to yield thecorresponding persilylated lactam (0.533 g, 93%): Rf=0.20 (hexane:EtOAc7:1); ¹ H-NMR δ (CDCl₃) 6.11 (s, 1 H, broad) 4.3 (d, 1 H, J=2.7 Hz),3.76 (dd, 1 H, J=4.5, 2.7 Hz), 3.7 (m, 1 H), 3.56-3.51 (m, 2 H), 3.26(m, 1 H), 0.12-0.0 (m, 36 H); IR (CHCl₃), 3390, 2970, 1675, 1460, 1310,1250, 1100 cm⁻ 1 ; ¹³ C-NMR δ (CDCl₃) 170.5, 74.9, 69.3, 68.8, 64.1,60.6, 0.10, 0.05, -0.21, -0.87. To a solution of the persilylatedmannolactam (176 mg, 0.378 mmol) in benzene (12 mL) under Ar was addedLawesson's reagent (136 mg, 0.337 mmol) and the suspension was heated toreflux for 1 h. After concentrating the homogeneous reaction mixture invacuo, the residue was suspended in CH₃ OH (10 mL), acidified [8 dropsfrom a solution of CH₃ OH (10 mL) and AcCl (0.3 mL)] whereupon it becamehomogeneous after 90 minutes at room temperature. Concentration affordeda white solid (230 mg) which was triturated with CHCl₃ (30 mL in 2 mLportions). The residue (81.2 mg) was purified by SiO₂ chromatography ona 5 inch×15 mm column eluting with CH₃ CN:H₂ O:AcOH 200:4:1 (3 mLfractions) to afford D-mannothionolactam (38 mg, 53%): R_(f) =0.30 (CH₂Cl₂ CH₃ OH:NH₄ OH 7:3:1); [α]_(D) +53.3° (c=0.69, MeOH); ⁻¹ H-NMR δ (D₂O) 4.29 (d, 1 H, J=3.7 Hz), 3.94 (dd, 1 H, J=3.8, 5.4 Hz), 3.8 (dd, 1 H,J=12.0, 3.6 Hz), 3.77 (dd, 1 H, J=5.3, 7.1 Hz), 3.66 (dd, 1 H, J=5.6,12.0 Hz), 3.32 (m, 1 H); ¹³ C-NMR δ (D₂ O, dioxane ref.) 202.8, 72.7,72.0, 67.7, 60.9, 60.5; IR (KBr) 3390, 2910, 1620, 1540, 1390, 1120,1060; CIMS 194 (M+1, 100%); EI-MS 193 (M⁺, 100%), 157 (96%), 140 (29%),139 (26%), 111 (54%), 102 (32%).

Preparation of D-Mannoamidrazone

Anhydrous NH2NH2 (60 μL, 1.89 mmol, distilled from NaOH) was addeddropwise to a stirred solution of the D-manno-thionolactam above (14 mg,0.072 mmol) in anhydrous CH₃ OH (3 mL) in an ice-H₂ O bath under Ar. TLCmonitoring (CH₃ CN: AcOH:H20 20:1:4) indicated that the startingmaterial disappeared in 90 minutes. The solution was concentrated invacuo and the residue (16 mg) was purified by SiO₂ chromatography (17mm×6 cm column; 25:3:1 CH₃ CN:H₂ O:AcOH 2 mL fraction) to affordD-mannoamidrazone

HOAc (14 mg, 75%): R_(f) =0.35 (CH₃ - CN:H₂ O:AcOH 10:4:1); [α]_(D)+10.9° (c=0.46, CH₃ OH); ¹ H-NMR δ (D₂ O) 4.64 (d, 1 H, J=3.4 Hz), 3.97(dd, 1 H, J=3.6, 4.8 Hz), 3.89 (dd, 1 H, J=4.9 Hz), 3.78 (dd, 1 H,J=4.5, 11.8 Hz) 3.67 (dd, 1 H, J=5.9, 11.8 Hz), 3.37 (m, 1 H), 1.84 (s,3 H); ¹³ C-NMR δ (D₂ O, dioxane ref.) 179.1, 164.1, 70.8, 67.5, 64.7,61.1, 58.2, 22.1; IR (KBr) 3290, 2930, 1705, 1575, 1410, 1350, 1120,1065, 101 cm⁻¹.

High Resolution FAB-MS: Calc. for C₆ H₁₄ O₄ N₃ : 192.0984; Found:192.0980.

EXAMPLE 10 Preparation of D-Galacto-N,N-Dimethylamidrazone

The procedure of Example 4 is repeated to produce D-galactothionolactamwhich is reacted with 1,1-dimethylhydrazine as in Example 8 to produceD-galacto-N,N-dimethylamidrazone.

EXAMPLE 11 Preparation of Riboamidrazone

The procedure of Example 8 is repeated to produce D-ribothionolactamwhich is reacted with hydrazine as in Example 8 to produceD-riboamidrazone.

EXAMPLE 12 Preparation of D-Glucoamidrazone-N-Phenylurea

The procedure of Example 8 is repeated except that the hydrazine isreplaced by an equivalent amount of 4-phenylsemicarbazide (NH₂-NH-CONHPh and the solvent is acetonitrile. Reaction readily occurs toproduce the D-glucoamidrazone-N-phenylurea.

EXAMPLE 13 Preparation of D-Glucoamidoxime Preparation ofD-Glucoamidoxime

Anhydrous hydroxylamine (200 μL of a 1.25M CH₃ OH solution) was addedunder Ar to a stirred solution of D-gluconothiolactam (10 mg, 0.052mmol) in CH₃ OH (2.5 mL). After 14 hours at room temperature, the lactamwas completely consumed and a new spot had appeared at Rf 0.14 (CH₂ Cl₂CH₃ OH:NH₂ OH 7:3:1). The solution was concentrated in vacuo and theresidue (17 mg) was purified by SiO₂ chromatography (3"×16 mm column;CH₃ CN:H₂ O:HOAc 30:3:1, 2 mL fractions) to afford D-glucoamidoxime HOAc(10 mg, 75%): Rf 0.41 (CH₃ CN:H₂ OHOAc 10:4:1); [α]_(D) +62° (c=0.39,CH₃ OH); ¹ H-NMR δ (D₂ O) 4.49 (d, 1 H, J=10.1 Hz), 4.18 (dd, 1 H, J=2.2Hz), 3.86 (dd, 1 H, J=2.3, 10.1 Hz), 3.69 (m, 3 H), 1.83 (s, 3 H); ¹³C-NMR δ (D₂ O, CH₃ OH ref.) 179.1, 156.4, 74.3, 68.4, 60.9, 57.8, 22.0,;IR (KBr) 3400, 2920, 1660, 1590, 1570, 1420, 1335, 1110, 1020 cm⁻¹.

High Resolution FAB-MS: Calc. for C₆ H₁₃ O₅ N₂ : 193.0824; Found:193.0821.

EXAMPLE 14 Preparation of D-Gluco-N-Methylamidoxime

The procedure of Example 13 is repeated except that the hydroxylamine isreplaced by an equivalent amount of N-methylhydroxylamine to produce theD-gluco-N-methylamidoxime.

EXAMPLE 15 Preparation of D-Mannoamidoxime

Anhydrous hydroxylamine (prepared as above, 280 μL) was added under Arto a stirred solution of mannonothiolactam IX (12.2 mg, 0.063 mmol) inCH₃ OH (2.5 mL). After 24 hours at room temperature, the D-mannolactamwas completely consumed and a new spot had appeared at Rf 0.10 (CH₂ Cl₂:CH₃ OH:NH₂ OH 7:3:1). The solution was concentrated in vacuo and theresidue (16 mg) was purified by SiO₂ chromatography (2"×16 mm column:CH₃ CN:H₂ O:HOAc 30:3:1, 1.5 mL fractions) to afford D-mannoamidoximeHOAc (11.6 mg, 73%): Rf 0.60 (CH₃ CN:H₂ O:HOAc 10:4:1); [α]_(D)-1.0.(c=0.40, CH₃ OH); ¹ H-NMR δ (D₂ O) 4.64 (d, 1 H, J=2.2 Hz), 3.38(m, 1 H), 1.95 (s, 3 H); 13C-NMR δ (D₂ O, CH₃ OH ref.) 178.8, 156.0,71.6, 66.5, 66.4, 61.7, 58.1, 21.8,; IR (KBr) 3390, 2930, 1660, 1560,1545, 1420, 1340, 1100, 1055, 1010 cm⁻¹.

High Resolution FAB-MS: Calc. for C₆ H₁₃ O₅ N₂ : 193.0824; Found:193.0825.

EXAMPLE 16 Preparation of L-Idoamidoxime

The procedure of Example 5 is repeated to produce L-idothionolactamwhich is reacted with hydroxylamine as in Example 12 to produceL-idoamidoxime.

EXAMPLE 17 Preparation of D-Glucoamidoxime-N-Phenylurethane

The D-glucoamidoxime of claim 13 is reacted with phenyl isocyanate inacetonitrile at a temperature ranging from 0° C. to room temperature tosubstantially quantitatively produce theD-glucoamidoxime-N-phenylurethane.

GENERAL BIOLOGICAL PROCEDURES A. Preparation of Solutions for EnzymeAssays

Phosphate Citrate Buffers--Phosphate-citrate buffers at constant ionicstrength (0.5M) were prepared according to a published procedure.(2)

HOAc/NaOAc Buffer--To an aqueous solution of acetic acid (0.4M, 100 mL)was added aqueous sodium acetate (0.4M, 100 mL). The resulting solutionwas adjusted to pH 5.0 by addition of aqueous sodium hydroxide (6.0M).

NaCl/NaOAc Buffer--A mixture of aqueous NaCl solution (0.1M, 2.0 mL) andHOAc/NaOAc buffer (0.75 mL) was diluted with deionized water (17.25 mL)to yield NaCl/NaOAc buffer (pH 5.0).

Glycine Buffer--Glycine (22.47 g, 0.299 mol) was dissolved in deionizedwater (705 mL, final concentration: 0.42M). The resulting solution wasadjusted to pH 10.4 by aqueous NaOH (6.0M).

pH for Enzyme Assays--Yeast α-glucosidase and green coffee beanα-galactosidase were assayed at pH 6.6. Almond β-glucosidase andjackbean α-mannosidase were assayed at pH 5.0. Bovine liverβ-galactosidase was assayed at pH 7.0.

Working Enzyme Solutions--Typical enzyme concentrations in the assaybuffer for inhibitor screening were as follows: yeast α-glucosidase, 10μL of enzyme suspension in 2.00 mL pH 6.6 phosphate-citrate buffer;almond β-glucosidase, 25 μL of enzyme solution (0.4 mg solid enzyme in70 μL pH 5.0 NaCl/NaOAc buffer) in 5.00 mL pH 5.0 NaCl/NaOAc buffer;jackbean α-mannosidase, 5-8 μL of enzyme suspension in 2.00 mLNaCl/NaOAc buffer; green coffee bean α galactosidase, 15 μL of enzymesuspension in 1.2 mL NaCl/NaOAc buffer; bovine liver β-galactosidase,1.7 mg solid enzyme in 850 μL NaCl/NaOAc buffer.

B. General Assay Procedure

An acceptable concentration of enzyme, upon hydrolysis of thecorresponding glycoside, should give an absorbance reading between 0.4and 2.0 at 400 nm. A control assay was first performed to determinewhether the enzyme concentration was acceptable. The assays involvedaddition of working enzyme solution (0.09 mL) and deionized water (0.18mL) to buffer at the appropriate pH (0.09 mL, phosphate-citrate). Theresulting solution was incubated at 37° C. for 5 minutes. Aliquots (0.1mL) of this preincubation mixture was then added to three separate testtubes. Substrate (the corresponding p-nitrophenyl-D-glycoside, 0.1 mL,10 mM in appropriate buffer), which had also been incubated at 37° C.,was then added to each tube at thirty-second intervals. The hydrolyseswere allowed to continue for 0.25 hours and then quenched by addition ofglycine buffer (2.5 mL). The absorbance of the resulting solution wasmeasure at 400 nm. Once this control run of the system gave anabsorbance within 0.4-2.0, the potential inhibitors were assayed againstthis working enzyme solution at final inhibitor and substrateconcentrations of 1 and 5 mM, respectively. In the inhibitor assays,deionized water was replaced by the inhibitor (4 mM solution indeionized water). All inhibitors were tested in triplicate, with acontrol (by substituting the inhibitors with deionized water) and astandard.

C. General Procedure for K_(I) Determination

Five substrate solutions (usually 2-20M) and five inhibitor solutions(usually 0-200 μM) were prepared. To a -mL test tube were added bufferat the appropriate pH (0.28 mL, phosphate-citrate), working enzymesolution (0.28 mL) and inhibitor solution (0.56 mL). The solution wasincubated at 7.C. for 5 minutes. Aliquots (0.1 mL) of this solution werethen pipetted into ten separate test tubes. Substrate solution (0.1 mL)was then added to each tube at thirty-second intervals. Duplicatereactions were terminated by addition of glycine buffer (2.5 mL) at theend of 3, 6, 8, 10, and 12 minutes. The amount of releasedp-nitrophenolate was measured spectrophotometrically at 400 nm. Theprocess was repeated for all combinations of inhibitor and substrateconcentrations.

The velocities (V) of substrate hydrolysis at every combination ofinhibitor [I] and substrate [S] concentrations were determined byplotting the absorbance values with respect to time and then calculatingthe slopes of the lines. Double reciprocal plots of 1/V versus 1/ [S] atdifferent [I] were then generated. The slopes of each of these lineswere then plotted against [I] and the data were fitted to a straightline. The [I]-intercept gave the enzyme-inhibitor dissociation constant.K_(I) values were also calculated from Hanes-Woolf plots of [S]/V vs.[S] by replotting the [S]/V intercepts versus [I] and ascertaining the[I] intercept. K_(I) values in the Table represent an average of the twocalculations.

EXAMPLE 18 Evaluation of Inhibitory Effects

In accordance with the above General Biological Procedures various ofthe above prepared amidines, amidrazones, and amidoximes were evaluatedto determine their glycosidic enzyme inhibitory capacity.

A summary of the results of the assays are provided in Table I below inwhich the compounds which were tested are identified by the exampleabove by which they were prepared and, in some cases, in graphical formin FIGS. 1-11. In the Table, "potent" means a K_(I) of less than about100 μM, "mod" is moderate which means a K_(I) of from about 100 to 1,000μM, and "weak" means a K_(I) of greater than about 1,000 μM.

                                      TABLE I                                     __________________________________________________________________________    Results of Bioassays in μM                                                        β-Glu                                                                          α-Glu                                                                       amylo-Glu                                                                           α-man                                                                          β-gal                                                                         α-gal                                Compound                                                                             pH 5.0                                                                              pH 6.6                                                                            pH 5.0                                                                              pH 5.0 pH 7.0                                                                             pH 6.6                                     __________________________________________________________________________    Nojirimycin                                                                          380   13  weak  moderate                                                                             weak mod-none                                   1       8    none                                                                              weak  9      potent                                                                             none                                       2       83   none                                                                              weak  potent potent                                                                             none                                       8       8.4 ± 0.9   6.5 ± 3                                                                           19                                              9      205 ± 25     0.166 ± 0.013                                                                     57 ± 2.5                                     13     13.8 ± 3     10 ± 2                                              15                       2 ± 0.1                                           __________________________________________________________________________     glu is almond glucosidase                                                     glu is yeast glucosidase                                                      amyloglu is A. niger amyloglucosidase                                         man is jackbean mannosidase                                                   gal is bovine galactosidase                                                   gal is coffee bean galactosidase                                         

COMPARATIVE EXAMPLE A

An attempt to prepare D-glucoamidine (Example 1) was made by aminolysisof the corresponding D-glucoiminoether, an alternative synthetic routestarting from D-glucolactam as in Example 1. The hydroxyl groups of thelactam were protected by being peracetylated (NaOAc, Ac₂ O, roomtemperature, 44 hr) to produce the tetra-O-acetyl lactam which was thentreated with Meerwein's salt (Borch, Tetrahedron Lett., 1968, 61-65)(triethyloxonium tetrafluoroborate, CH₂ Cl₂, room temperature, 36 hr) toproduce the D-glucoiminoether. Exposure of the iminoether to excessammonia in methanol regenerated the starting D-glucolactam in 72-84%yield. Attempted aminolysis by treatment with concentrated ammoniumhydroxide, anhydrous liquid ammonia, or ammonium chloride also producedthe starting lactam as the sole reaction product.

The re-formation of the lactam could be rationalized by postulatinginitial attack of ammonia on the C-3 acetate to generate an intermediatewhich could cyclize with loss of ethanol to form an acetamide ketalstructure which, upon prolonged exposure to methanolic ammonia wouldproduce the starting lactam. Thus an alternative hydroxyl protectinggroup, i.e. trimethylsilyl, which would not enter into suchneighboring-group participation was tried as described in Example 1.Attempts at preparing the D-glucoiminoether by O-alkylation wereunsuccessful. Instantaneous desilylation occurred when the blockedlactam was treated with triethyloxonium tetrafluoroborate, even whenbuffered with Na₂ HPO₄.

Other literature procedures for O-alkylation of lactams to produce animinoether (ethyl chloroformate (Suydam et al., J. Org. Chem., 1969, 34,292-6), dimethylsulfate (Benson, et al., In Organic Syntheses CollectiveVolume IV, Rabjohn, Ed., John Wiley: New York, 1963, pp 588-590),diazomethane (Nishiyama et al., Tetrahedron Lett., 1979, 48, 4671-4))also failed to produce the corresponding iminoether.

EXAMPLE 19 Alternative Preparation of D-Mannoamidine

The procedure of the first paragraph of Comparative Example A wasrepeated but starting with the iminoether of D-mannose rather than ofD-glucose. Specifically, to a solution of D-mannoiminoether (27 mg,0.072 mmol, 1.0 equiv) in methanol at -25° C. was added NH₃ -MeOHsolution (4.6M, 3.5 mL). The resulting solution was stirred at -25° C.for 24 hr, at -5° C. for 24 hr, and finally at room temperature for 12hr. The solution was concentrated in vacuo and chromatographed (7:3:1CH₂ Cl₂ :CH₃ OH:NH₄ OH) to afford D-mannoamidine.

Various other examples will be apparent to the person skilled in the artafter reading the present disclosure without departing from the spiritand scope of the invention. It is intended that all such other examplesbe included within the scope of the appended claims.

What is claimed is:
 1. The compound D-glucoamidoxime.
 2. The compoundD-gluco-N-methylamidoxime.
 3. The compound D-mannoamidoxime.
 4. Thecompound L-idoamidoxime.